Normal Patterns of Spontaneous Activity Are Required for Correct Motor Axon Guidance and the Expression of Specific Guidance Molecules  M.Gartz Hanson, Lynn T. Landmesser  Neuron  Volume 43, Issue 5, Pages 687-701 (September 2004) DOI: 10.1016/j.neuron.2004.08.018

Figure 1 Effect of Chronic In Ovo Drug Treatments on the Frequency of Spontaneous Rhythmic Bursting Episodes (A) At St 23, rhythmic bursts are recorded from ventral root fascicles. (B and C) The interval between these bursts in isolated cord preparations (B) and the interval between in ovo axial movements (C) are significantly slowed by picrotoxin (#p < 0.05). (D–F) Bar graphs showing the interval between spontaneous bursting episodes between St 25 and 30 in embryos chronically treated with picrotoxin, strychnine, or combined picrotoxin/strychnine from St 20 until the age sacrificed. Each graph shows values for controls (open bars), drug-treated embryos dissected in the continued presence of the drug (black bars), and drug-treated embryos following drug washout (gray bars). Complete blockade of bursting is denoted by asterisks. (G) Suction electrode recordings from crural nerves from a St 25 chronic picrotoxin-treated embryo, following dissection in 50 μM picrotoxin (top trace) and upon drug washout. (H) Similar recordings from an embryo chronically treated with strychnine. (I) The frequency of such unit activity occurring between episodes was significantly greater (#p < 0.05) following chronic picrotoxin treatment. (J and K) Spontaneous single-unit activity (arrowheads) occurring between rhythmic episodes in St 23 control (J) or chronic picrotoxin-treated embryos (K). Neuron 2004 43, 687-701DOI: (10.1016/j.neuron.2004.08.018)

Figure 2 The Location of Retrogradely Labeled Motoneuron Somata in St 30 Control and Chronically Drug-Treated Embryos Cross-sections from the crural spinal cord showing the location of motoneurons labeled following HRP injection into the sartorius (A–C), the femorotibialis (D–F), and adductor (G–I) muscles. The lateral motor column and its medial and lateral subdivisions are indicated by the dotted white line. Arrows indicate misplaced somata not observed in control (A, D, and G) or chronic strychnine-treated (C, F, and I) embryos. Histograms below each section are examples of the anterior-posterior location of all of the appropriately (gray bars) or inappropriately (black bars) located somata in single control or drug-treated embryos. Calibration bar, 100 μM. Neuron 2004 43, 687-701DOI: (10.1016/j.neuron.2004.08.018)

Figure 3 Characterization of the LIM Transcription Factor Code of St 30 Retrogradely Labeled Motoneurons in Control and Picrotoxin-Treated Embryos In control embryos (A, B, and C), all motoneurons labeled from the adductor muscle were in the medial division of the lateral motor column (LMCm) and were Lim1/2 (green) negative (A), while those labeled from the sartorius muscle were in the lateral division of the lateral motor column (LMCl) and were Islet1/2 negative ([B]; red) and Lim1 positive (C). Following chronic picrotoxin treatment, motoneurons located out of their normal pool location following HRP labeling from the adductor (D) or sartorius (E and F) muscles are enclosed in a yellow box and shown at higher magnification in (G), (H), and (I). Following HRP injection of the adductor, motoneurons located in an inappropriate lateral position (G) express Lim1/2 (green), consistent with a LMCl identity. Medially misplaced motoneurons labeled from the sartorius (H) express Islet1/2, consistent with a LMCm identity. (I) Several motoneurons labeled from the sartorius are located in the position of the medial motor column and are Lim1/2 negative. Calibration bar, 100 μM in (A)–(F) and 20 μM in (G) and (H). In (A)–(F), the outline of the lateral motor column and its lateral and medial divisions are shown by the white dotted line. Neuron 2004 43, 687-701DOI: (10.1016/j.neuron.2004.08.018)

Figure 4 The Behavior of St 23–24 Axons in the Plexus Region in Control and Picrotoxin-Treated Embryos The trajectories of individual enhanced GFP-labeled motor axons were reconstructed from thick vibratome sections cut transverse to the cord (examples shown at top) using Neurolucida software after in ovo electroporation of the GFP construct under the control of the HB9 promoter at St 16. Below are shown a number of such trajectories, individually color coded and superimposed, for St 23.5 control (A), St 24 control (B), and St 24 chronic picrotoxin treatment (C). Calibration bar, 100 μM. Neuron 2004 43, 687-701DOI: (10.1016/j.neuron.2004.08.018)

Figure 5 The Expression of PSA-NCAM on LMCl and LMCm Motoneurons Following Chronic Picrotoxin or Strychnine Treatment (A), (D), and (G) show immunostaining with an antibody that recognizes all isoforms of NCAM, and (B), (E), and (H) show immunostaining for PSA-NCAM in transverse limb sections at the level of the plexus. The color panels at right show the merged NCAM (green) and PSA-NCAM (red) images. In St 25 control (A–C) and strychnine-treated (G–I) embryos, PSA is expressed more strongly on dorsal than on ventral (arrows) axons but is downregulated on both by picrotoxin treatment (D–F). (J and K) Cross-sections of St 25 control and picrotoxin-treated spinal cords, immunostained for PSA (top) and NCAM (bottom). Following picrotoxin treatment, PSA is downregulated in the LMC that contains the motoneuron somata, but its expression on commissural axons (arrows) and on cells in the myotome (asterisks) is not affected. (L)–(N) show bar graphs quantifying PSA levels on dorsal and ventral axons relative to control dorsal and ventral axons for St 24, 25, and 29, respectively. At both St 24 and 25, PSA is significantly reduced (asterisks) from control values following picrotoxin (p < 0.05) but not strychnine treatment. (O and P) Similar quantification for the expression of CRYPα at St 24 and 29. (Q) Following sarcosine treatment, PSA levels were not altered significantly. Calibration bar, 200 μM for (A)–(I) and 100 μM for (J) and (K). Neuron 2004 43, 687-701DOI: (10.1016/j.neuron.2004.08.018)

Figure 6 EphA4 Expression on St 24–29 Motoneurons (A), (D), and (G) show immunostaining for neurofilament 160 (NF-160), while (B), (E), and (H) show EphA4 immunostaining for St 25 axons, in sections transverse to the limb at the divergence of the dorsal and ventral nerve trunks, in control or picrotoxin- or strychnine-treated embryos. Color panels at right show the merged images of NF-160 (green) and EphA4 (red). In St 25 control (A, B, C, and O) and strychnine-treated (G, H, I, and O) embryos, EphA4 is expressed on dorsal but not ventral axons (arrows). Chronic picrotoxin treatment resulted in downregulation of EphA4 from dorsal axons at St 25 (D, E, F, and O) and St 24 (N). St 28 and 29 control embryos still exhibit the differential D-V staining for EphA4, expressed as a percent of control values (P and Q). Chronic picrotoxin-treated embryos have now begun to upregulate, while those treated with strychnine have downregulated EphA4 on dorsal axons. Asterisks indicate values that differ significantly from control values (p < 0.05). (R) Increasing the frequency of spontaneous episodes with sarcosine treatment does not affect EphA4 expression. (J and K) Cross-sections of spinal cord from St 24 control and picrotoxin-treated embryos immunostained for EphA4. (L) and (M) (left panels) show corresponding levels of EphA4 mRNA at St 24 revealed by in situ hybridization, while right panels show Lim1 and Islet1 immunostaining of the same sections. LMCs are encircled by dotted lines. Calibration bar, 200 μM for (A)–(I) and 100 μM for (J)–(M). Neuron 2004 43, 687-701DOI: (10.1016/j.neuron.2004.08.018)

Figure 7 The Effect of Blocking Spontaneous Rhythmic Bursting from St 25 to 30 with Combined Strychnine/Picrotoxin Treatment on the Expression of Islet1 and Lim1 Panels show immunostaining for Lim1 (green; [E and F]) and Islet1 (red; [G and H]) or both (A–D) in transverse sections of lumbar spinal cord. Medial is left; dorsal is up. The LMC is encircled by a dotted line. Asterisks indicate the location of the adjacent dorsal root ganglion. The control pattern of Lim1/2 and Islet1/2 staining at St 25 and 26 (A and C) was not altered following combined picrotoxin and strychnine treatment (B and D). However, by St 30 Lim1/2 was essentially absent from the LMCl cells (F) compared to control (E), where robust staining was evident in the lateral cells. Compared to control (arrowhead in [G]), Islet1/2 was also strongly downregulated from the medial motoneurons (H), although some weakly stained cells (arrowhead) were still present. Downregulation of Islet1/2 on dorsal root ganglion cells (asterisks in [G] and [H]) did not occur, but downregulation of these factors in lumbar interneurons did. Calibration bars, 100 μM. Neuron 2004 43, 687-701DOI: (10.1016/j.neuron.2004.08.018)